DNA Nanotags: Bright Fluorescent Labels and Sensors for Intracellular Imaging Understanding the molecular basis for human disease is essential for developing effective therapeutics with minimal side effects. The fundamental biological processes underlying both healthy and diseased states involve transient intermolecular (e.g. protein-protein or protein-nucleic acid) interactions within and at the surface of cells. Direct imaging of these interactions in real time provides unparallelled insight into the affinity and kinetics of molecular association. The overwhelming majority of such experiments are done using fluorescence microscopy and fusion constructs between proteins of interest and green fluorescent protein (GFP) or other FPs. While substantial progress has been made with these fusion proteins, the relatively low brightness and photostability of GFP hinder applications that require either short imaging times or high sensitivity due to low abundance of the protein of interest. The main objective of this proposal is to create a new class of bright fluorescent labels that will exhibit greatly improved brightness and photostability relative to GFP. This will be accomplished by synthesizing polychromophore assemblies consisting of a branched DNA nanostructure with dozens of covalently attached intercalating dyes. The design of these DNA nanotags takes advantage of 50 years of knowledge concerning the use of fluorescent intercalating dyes for the detection of DNA as well as more recent work in the design and synthesis of DNA nanostructures. The high brightness of the nanotags derives from their very large effective extinction coefficients due to the presence of many dyes bound to each DNA scaffold. Attachment of additional longer wavelength dyes to the DNA termini will lead to efficient energy transfer and tuning of the fluorescence color throughout the visible and near-IR regions of the spectrum. The nanotags will be optimized in terms of their biochemical and photochemical stability through rational design of the dye and DNA structures. Finally, nanotag-antibody conjugates will be synthesized and tested for labeling of yeast cell surfaces and within Drosophila embryos. Overall, this proposal combines organic chemistry, single- molecule spectroscopy and fluorescence microscopy to create a new class of generally useful, bright fluorescent labels. Project Narrative DNA Nanotags: Bright Fluorescent Labels and Sensors for Intracellular Imaging The proposed research will lead to a new class of fluorescent labels that will be available in virtually any color and can be attached to various recognition modules to allow staining of cell surface and intracellular targets present at very low concentration. The ability to detect and track single molecules in cells will significantly advance our understanding of fundamental biological processes and the molecular-level distinctions between healthy and diseased states, ultimately allowing development of more potent therapeutics with fewer side effects.